1. Field of the Invention
This invention involves a method for measuring the quantity of analytes in biological samples, and, more particularly, a method for measuring the quantity of analytes in biological samples by means of deactivation of a chemiluminescent label.
2. Discussion of the Art
Current assays for blood alcohol level are based on reactions catalyzed by two enzymes, yeast alcohol dehydrogenase and diaphorase, followed by attenuation of the light emitted by a fluorophore. These assays are often referred to as "radiative energy attenuation" assays, or REA assays. REA assays involve color development reactions. Their reaction systems use analyte to convert a chromogen (unreacted dye) to a chromophore (colored dye). A stable fluorescent substance (fluorophore) is also included in the reaction mixture. The light-absorbing properties of the chromophore produced cause a decrease of measured fluorescent light intensity from the fluorophore. REA assays are used quantitatively to measure specific analytes based on the principle that the logarithm of measured fluorescent light intensity is inversely proportional to the amount of chromophore present. Production of chromophore is linked by the reaction system to consumption of analyte, so development of fluorescent attenuation can be calibrated to measure the concentration of analyte in the sample. The reactions for determining ethanol concentration in a biological sample can be expressed as follows: ##STR1## where yADH represents yeast alcohol dehydrogenase;
NAD.sup.+ represents nicotinamide adenine dinucleotide; PA2 NADH represents the reduced form of nicotinamide adenine dinucleotide; PA2 MTT represents 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; and PA2 MT-Formazan represents a light absorbing compound (i. e., a chromophore).
In the foregoing reactions, the reaction product MT-Formazan blocks the transmission of light. The greater the quantity of MT-Formazan formed in the foregoing reaction, the greater is the quantity of ethanol in the sample. The foregoing reactions are employed to determine the concentration of ethanol by means of an assay employing an AxSYM.RTM. instrument, commercially available from Abbott Laboratories. The relationship between the concentration of ethanol and the measured fluorescence intensity is established by generating a calibration curve. Ethanol calibrators of known concentration are run and the resulting attenuated fluorescence signal is measured. When an unknown is read, its concentration is calculated from the stored calibration curve. The measurement arrangement for determining the concentration of ethanol is illustrated schematically in FIG. 1.
The quantitative measurement of ethanol in biological samples has so far relied upon the following general methods:
(1) chemical oxidation of ethanol in the presence of various oxidizing agents; PA1 (2) biochemical oxidation of ethanol catalyzed by alcohol dehydrogenase enzymes and subsequent colorimetric measurement of NADH; PA1 (3) biochemical oxidation of ethanol catalyzed by alcohol dehydrogenase enzymes followed by the formation of formazan in the presence of diaphorase; formazan then attenuates the light emitted by a fluorescent compound, which is a reagent critical to the method. PA1 (a) combining the biological sample, at least one oxidizing enzyme for the analyte of interest, nicotinamide adenine dinucleotide (hereinafter NAD.sup.+), and a chemiluminescent label to form a reaction mixture; PA1 (b) allowing the analyte to undergo an oxidation-reduction reaction and NAD.sup.+ to be converted to the reduced form of nicotinamide adenine dinucleotide (hereinafter NADH) and further allowing the chemiluminescent label to react with NADH; and PA1 (c) determining the concentration of the analyte of interest in the biological sample by correlating the quantity of light emitted with the concentration of NADH. PA1 (a) combining the biological sample, a solid phase, at least one oxidizing enzyme for the analyte of interest, nicotinamide adenine dinucleotide (NAD.sup.+), and a chemiluminescent label to form a reaction mixture; PA1 (b) allowing the analyte to undergo an oxidation-reduction reaction and NAD.sup.+ to be converted to the reduced form of nicotinamide adenine dinucleotide (NADH)) and further allowing the chemiluminescent label to react with NADH; PA1 (c) separating the chemiluminescent label from the solid phase; and PA1 (d) determining the concentration of the analyte in the biological sample by correlating quantity of light emitted with the concentration of NADH. PA1 (a) combining a biological sample, a solid phase, at least one oxidizing enzyme for the analyte of interest, and NAD.sup.+ to form a reaction mixture; PA1 (b) allowing an analyte, e. g., ethanol, to be converted to a bio-oxidation product, e. g., acetaldehyde in the case of ethanol, and NAD.sup.+ to be converted to NADH; PA1 (c) adding a chemiluminescent label to the reaction mixture; PA1 (d) allowing the chemiluminescent label to react with NADH and be captured by the solid phase; PA1 (e) washing the reaction mixture; PA1 (f) releasing the chemiluminescent label; and PA1 (g) determining the concentration of analyte by correlating quantity of light emitted with the concentration of NADH.
These methods suffer from various disadvantages. Assays employing chemical oxidation of ethanol exhibit low sensitivity and are primarily used to provide a preliminary estimate of alcohol intoxication. Assays employing the second method exhibit fairly low sensitivity as well and are susceptible to interference from other light absorbing substances in a sample. Assays employing the third method require a source of fluorescence and a corresponding detection system. Accordingly, a more robust and cost-effective method for quantitative determination of ethanol in biological samples is needed.
U.S. Pat. No. 4,950,613 discloses a method of preparing a labelled specific binding partner, such as a biological probe in the form of an antibody or oligonucleotide probe, using a protected label (the corresponding unprotected label being susceptible to inactivation, such as by hydrolysis, to yield a non-chemiluminescent form of the label). The specific binding partner is linked to the label, and an adduct of the label is prepared using a protective adduct former, which produces a protected label, which is less susceptible to inactivation. Particularly preferred are the acridiniums and acridans. Formation of the protected label is preferably an equilibrium reaction that is readily reversible, such as by dilution or oxidation of the protective adduct former.
U.S. Pat. No. 5,294,540 discloses a multilayer analytical element for quantitatively assaying ethanol comprising a tetrazolium salt, alcohol dehydrogenase, NAD.sup.+, and an electron transfer agent. The layer comprising the electron transfer agent also includes a polymer having recurring negatively charged groups, and the NAD.sup.+ is in a different layer.
U.S. Pat. No. 5,624,813 discloses chemiluminescence-based assays that detect or quantify NAD(P)-linked dehydrogenases and oxidoreductases, or the cofactors, or detect or quantify substrates, intermediates or products of reactions catalyzed by these enzymes by coupling the enzyme reactions to luminescence generating systems. The assays include the steps of reacting a peroxidase with the NAD(P)H produced in a reaction catalyzed by an oxidoreductase that requires NAD(P).sup.+ /NAD(P)H as a cofactor; and then adding a chemiluminescent moiety to produce chemiluminescence from which the analyte, such as an amino acid or sugar, the activity of the oxidoreductase or NAD(P).sup.+ /NAD(P) analyte is determined.
Up to now, no assay for ethanol has effectively eliminated or reduced the interference from substances present in the biological sample. An example of such interference is background absorption in colorimetric assays.